Cellular and Molecular Serum levels of immunoglobulin and complement in UTI of patients caused by Proteus mirabilis and using AgNPs as antiswarming

: The use of plant extracts represents a promising approach for the synthesis of silver nanoparticles (AgNPs). This study reports the low-cost, green synthesis of AgNPs using the extract of clove and black seeds. The biosynthesized AgNPs were confirmed and characterized by analysis of the spectroscopy profile of the UV-visible spectrophotometer. The purpose of the present study is to evaluate the inhibitory effect concentration (MIC) of AgNPs, clove, and black cumin seed extracts on the growth and swarming of P. mirabilis . Clinical isolates of P. mirabilis were isolated from patients suffering from urinary tract infections. Thir teen types of antibiotics were used in the present study to detect their ability to inhibit P. mirabilis 's resistance. Immunological findings included the determination of serum levels of IgG, IgM, IgA and complement protein C3 and C4. Results showed that IgG and IgA concentrations significantly increased (1311.13 ± 72.54 and 279 ± 21.31) respectively in UTI patients in comparison to the healthy control group which was 1089.88 ± 37.33 and 117.611 ± 4.19 respectively, While IgM concentrations were increased non significantly in UTI patients (153.331 ± 6.45) in comparison to healthy control (145.2 ± 13.49). Complement components C3 showed a significant increase in UTI patients with mean values of 125.95 ± 6.22 compared to the control group with mean values of 55.191 ± 9.64, while C4 showed statically non-significant among UTI patients in comparison with the control group (35.195 ± 2.34 and 34.371 ± 1.22) respectively .


Introduction
Urinary tract infections (UTI) are mostly of the ascending type, hence, urinary tract mucosa including urinary immunoglobins (secretory IgA) is considered as the frontline of defense against bacterial infection. Local immunoglobulin of the urinary tract serves as defending agents that inhibit bacterial attachment to mucosal surfaces (1). Serum antibodies found in UTI-positive patients have bactericidal activity (2). Antibodies in urine may resist UTI by preventing the adherence of bacteria to uroepithelial cells, the role of humoral immune response which increased IgG and IgA, IgM production were associated with UTI. Also, bacteria are killed by infected human serum through the lytic activity of the complement system. Complement 3 (C3) levels are significantly higher in the urine of UTI patients and uropathogenic E. coli may stimulate C3 production (3). UTIs are one of the human infectious diseases caused by a bacterial infection that affects any part of the urinary tract (4). UTI represents one of the most common types of infections in the human body. Various bacteria are involved in the development of UTI, including Escherichia coli, Klebsiella, Pseudomonas, Proteus mirabilis and Enterococcus faecalis. It is the second most frequent kind of infection in the body. Bacterial organisms which cause UTI may involve Escherichia coli, Klebsiella, Pseudomonas, Proteus mirabilis and Enterococcus faecalis. less than 8% of uncomplicated UTIs is caused by Proteus mirabilis, a major pathogen often isolated from patients with complicated UTIs, i.e., those with functional or anatomic abnormalities of or with chronic foreign bodies in the urinary tract (5). As a motile gram-negative bacillus belonging to the family Enterobacteriaceae, P. mirabilis represents an opportunistic pathogen frequently found in intestinal tracts (6). P. mirabilis is considered a major cause of UTIs and the primary infectious factor in patients with indwelling urinary catheters (7). P. mirabilis can cause food poisoning, respiratory and wound infections, bacteremia, and other infections. P. mirabilis infection is due to the presence of a wide range of virulence factors, including the presence of extracellular proteins, motility of the pathogen by flagella, the activity of urease enzymes, binding to iron ions, invasion of cells, and so on. Lipopolysaccharides and hemolysins are also two important virulence factors that allow bacteria to attach to cells and colonize tissues, thus exacerbating pathogenesis. It should be noted, however, that the pathogenicity of this bacterium is not solely due to virulence genes; biofilm formation is also one of the main factors in the pathogenicity of P. mirabilis, which plays an important role in the persistence of infection and the occurrence of inflammatory reactions (6). After bacterial colonization, the catheter may enter the bladder, setting the stage for UTI (8).
Swarming and migration improve the ability of P. mirabilis to colonize the catheter and urinary tract. Swarming refers to the transformation of small, motile vegetative cells into multinucleated and aseptate swarms that are up to forty times larger than vegetative cells and have much larger flagella. These swarms migrate rapidly from the colony until they are paused (consolidated) and begin de-differentiation. The sequence of migration and consolidation cycles leads to the formation of larger colonies, which is characterized by the presence of concentric rings (9). It should be noted that biofilm formation plays a critical role in this migration (10). The transformation of vegetative cells into large, multinucleated and hyperflagellated swarms is one of the most important reasons for the retention of P. mirabilis pathogenicity. This is a banishment of the bacterial response to growth in viscous fluids. Swarm requires a lot of resources; in a way, a large part of the bacterial metabolism is allocated to the assembly and operation of the flagellum and other proteins involved in the swarm. This differentiation is due to the coordinated expression of the global regulon of 50 genes. These proteins contain a large set of virulence factors including flagellin, urease, hemolysin, and the ZapA metalloprotease degrading antibacterial peptides, such as defensin one and LL-37. Due to the coordinated expression of virulence factors during differentiation, it is believed that the transformation of vegetative cells into swarmer cells plays a key role in pathogenicity (11).
Nanoparticles represent a particle with a nanometer size of 1-100 nm. The nanoscale material has new, unique, and superior physical and chemical properties compared to its bulk structure due to an increase in the surface area's ratio per volume of the material/particle (12). Silver nanoparticles have found many applications in the medical industry due to their antibacterial properties and inhibitory effects on microbial growth (13). Due to the importance of silver nanoparticles, various methods such as chemical reduction, photochemical reduction, integrated methods, micro-emulsion radiation and green synthesis have been proposed to produce these particles (14). Recently, the use of herbal extracts has sometimes been proposed as a fast, cost-effective and environmentally friendly approach to the production of metal nanoparticles. By using such a green method, the toxicity of nanoparticles can be reduced and the problems related to chemical methods such as high cost and environmental pollution can be avoided. So far, many studies have shown that silver nanoparticles produced using plant extracts have antimicrobial and anticancer properties (15).
Swarming cell formation is an important feature of P. mirabilis infection. Swarming refers to the formation of bacterial concentric zones that cover the entire surface of the culture medium (8). Due to the occurrence of swarming, it is very difficult to isolate other bacteria from cultures containing P. mirabilis. At the same time, it should be borne in mind that swarming is a major problem for laboratory diagnostic activities. Therefore, the development of methods that can prevent the formation of swarms without harming other bacteria is clinically important. It is through swarming that bacteria can spread in the form of biofilms throughout the culture medium (16). This study is aimed to evaluate the effect of AgNPs of clove and black seed cumin on the swarming of P. mirabilis.

Sample collection and identification
Ethical approval for this research was confirmed by the scientific and ethics committee of Salahaddin University-Erbil of Iraq. A total of 283 samples from patients with UTI symptoms were screened, and finally, 51 isolates (18.02%) of P. mirabilis were isolated. The specimens were directly inoculated to Tryptone Soy Broth and streaked onto MacConkey agar and Blood agar plates and incubated aerobically at 37 °C for 24 hrs. The isolates were identified based on a series of microbiological and biochemical tests that routinely are used in microbiological diagnostic laboratories and confirmatory VITEK II system using (ID) GN cards (BioMérieux Inc. France). All positive cultures were undergoing the PCR assay for amplifying the ID gene to support the identification of P. mirabilis.

Molecular characterization of the isolates
For more confirmation of the identity of isolates, all P. mirabilis isolates were subjected to molecular characterization by detecting a specific gene or responsible to produce urease enzyme and regarded as a diagnostic feature of these bacteria using PCR.

Genomic DNA extraction
The confirmed DNA was used to amplify the ureR gene in P. mirabilis isolates. The sequence of the ureR gene was used to confirm the identity of P. mirabilis; short read sequence products of 225 bp length as amplicon size were produced with Z18752F 5´-ATA ATC TGG AAG ATG ACG AG-3' and Z18752R 5´-GGT GAG ATT TGT ATT AAT GG-3' (17). Briefly, PCR reaction of these genes was carried out in a total volume of 25 μL with the program: an initial denaturation at 94 °C for 4 mins, followed by 40 cycles, each consisting of denaturation at 94 °C for 40 sec; annealing at 58 °C for 1 min; extension at 72 °C for 20 sec, and a final extension at 72 °C for 10 mins. Then, the amplified samples were electrophoresed on agarose gel containing ethidium bromide to observe the PCR products.

Extraction of clove and black seed
A 10 g weight of powdered clove (18) and black cumin seeds (19) was suspended in 100 mL of sterilizing distilled water and shaken at a constant rotating speed of

Antibacterial potential analysis by agar disk diffusion method
The cultures of P. mirabilis inappropriate agar plates were conducted for the disk diffusion assay. Briefly, 100 μL of bacterial suspension ( ~1 × 10 8 CFU. mL -1 ), previously overnight cultivated in LB broth at 37 °C, was spread on the surface Mueller-Hinton agar plates. Then, five sterilized filter disks were placed on the agar plates conducted with 40 µL of the appropriate concentration of extracts and AgNPs at 45-50 °C. The plates were incubated at 37 °C (24 hrs.), and the relative clear zones of microbial inhibition were estimated in millimeters (mm). The antimicrobial activity of the extracts and AgNPs was determined from the clear zones of inhibition. For this purpose, the concentrations 512, 256, 128, 64, and 32 μg. mL -1 of the extracts and AgNPs were used to study disk diffusion. Gentamicin (10 μg) and DMSO (%5) were prepared as positive and negative controls, respectively (13).

The minimum inhibitory concentration (MIC)
The MIC and the minimal bactericidal concentration (MBC) of extracts and AgNPs were determined following Clinical and Laboratory Standards Institute (CLSI) guidelines. Bacteria cells were inoculated in the wells of a 96-microwell plate containing either LB broth alone (positive control) or LB broth containing AgNPs at a final concentration ranging and extracts performed the same protocol with different concentrations ranging from 32-512 µg. mL -1 . The microplates were incubated at 37 ⁰C for 24 hrs. at 250 rpm. The MIC value corresponded to the extracts and AgNPs doses that inhibited bacterial growth (compared to the positive control). MBC was also evaluated by sub-culturing the content of the first two clear wells obtained in the MIC assay onto LB agar plates (23). Amoxicillin (100 µg. mL -1 ) was used as the control for this bacterium. Optical density at 630nm (OD 630 ) was used to estimating the concentration of the bacterial population via ELISA reader (24).

Tolerance level
The tolerance level of tested bacterial strains against AgNPs and extracts was determined using the following formula (25): The characteristic of the antibacterial activity of AgNPs was determined by the tolerance level indicating the bactericidal or bacteriostatic action against the P. mirabilis. When MBC/MIC ratio is ≥16, the test agent's antibacterial efficacy is considered bacteriostatic, whereas MBC/MIC ≤4 indicates bactericidal activity.

Inhibition of the swarming phenomenon
Swarming motility experiments were performed as described by Aygül et al., 2019 (26). LB broth with 1.5% agar containing extracts and AgNPs at the required concentration were prepared. As a positive control, boric acid was used in swarming motility experiments. The plates could dry at 37 °C for 24 hours. After that, 1 mL of a fresh overnight culture of bacteria in LB broth was taken into a sterile microcentrifuge tube, and the cells were precipitated by centrifugation (12.000 ×g 150 rpm/min in a shaker for 48 hrs. It was centrifuged for 10 min at 4000 ×g and filtered through filter paper to get a clear extract. A concentration of 10.0 mg. mL -1 stock solution was prepared using distilled water via the serial dilution method.

Biosynthesis of silver nanoparticles
The synthesis of silver nanoparticles was achieved by dissolving AgNO 3 powder in distilled water to prepare a 10 mM stock solution (20). Mix extract with AgNO 3 (1:10 ratio) in the flask 250 mL and wrapped with an aluminum foil and was then heated in a water bath at ~2 hrs. at 90 ⁰C. The reaction mixture was adjusted to pH 10.0 by adding 1.0 M NaOH solution drop-by-drop to allow the magnetic precipitations to form uniformly. The reduction process of silver ions (Ag + ) to silver (Ag 0 ) nanoparticles was identified by the color change of solutions from yellow to brownish-yellow or deep brown depending on the reaction condition (21). After that, the AgNPs were obtained by repeated centrifugation at 10 000 ×g for 20 min, and then, discarded the supernatant and the pellet was washed using deionized water several times. The obtained samples were dried overnight in a vacuum oven at 60 °C and collected (20).

Characterization
UV-Visible absorption analysis was carried out by using a Shimadzu single-beam UV-Visible spectrophotometer in the range of 200-800 nm to study the successful formation of AgNPs. FT-IR spectroscopy analysis was carried out by using a Shimadzu 100 FTIR spectrometer in the range of 400-4000 cm -1 by crushing a small amount of sample AgNPs and plant extract with KBr powder to study the functional groups (22).

Ferric reducing antioxidant power (FRAP) assay
FRAP reagent was freshly prepared by the protocol of (15). Overall, 10 µL of extracts and AgNPs (0.1 mg. mL -1 ) was distributed into a 96-well plate, and then 200 µL of FRAP reagent was placed into the same well containing the sample. The solution was mixed and incubated for 30 min at 37 °C. The absorbance was recorded at 593 nm. A dark blue color formed as the Fe 3+ -TPTZ complex was reduced to Fe 2+ -TPTZ form by the anti-oxidant. The standard ascorbic acid curve was prepared for comparison using various concentrations, as shown in Figure 1. The following equation calculates the FRAP value for each compound. for 1 min). The supernatant was then discarded, 1 mL of 0.9% sodium chloride solution was added, and the residual bacteria were resuspended by vertexing. Five μL suspensions were added to the center of the dried plates. The plates were incubated reversely at 37 °C for 24 hrs. After 24 hrs., the diameter of the swarm circle was measured in millimeters and recorded. In triplicate experiments, the mean of swarm diameters detected on three plates was taken, and the results were analyzed compared with the negative control group.

Assessment of biofilm production of P. mirabilis
The biofilm formation by P. mirabilis was assessed according to Stefanovic 2000 with some modifications. Fresh tryptone soya broth (TSB) was added to an overnight culture of P. mirabilis to adjust its turbidity to 10 6 CFU. mL -1 . The wells of microplates were inoculated with aliquots of 200 μL of the adjusted bacterial suspension, and the plates were incubated for 24 h at 37 °C. The wells were gently aspirated and then washed thrice with sterile phosphate-buffered saline (PBS, pH 7.2) to remove any non-adherent cells. To fix the adherent cell, 200 μL of 99% methanol were added and left for 20 min. The wells were stained with 200 μL crystal violet (1%) for 20 min, and the unbound dye was removed under running distilled water and dried in air. The bound dye was eluted by adding 160 μL of 95% ethanol, and the optical densities of the stained adherent biofilms were read with a microplate reader at a wavelength of 490 nm. The test was repeated three times, and the average optical densities were calculated. The cut-off OD (ODc) that corresponds to three times standard deviations above the mean OD of the negative control was calculated, and the biofilm formation capacity was assessed as non-biofilm forming (OD ≤ODc), weak biofilm forming (OD > ODc, but ≤ 2ODc), moderate biofilm-forming (OD>2ODc, but ≤ 4ODc), or strong biofilm-forming (OD> 4ODc) ( Table 1) (27).

Assay of immunoglobulins and complements in serum
Venous blood samples (3 ml) were collected from UTI patients and control groups, sera were separated and stored at deep freeze until analysis of serum levels of immunoglobins (IgG IgA IgM) and complement (C 3, C 4) were estimated by using commercially available Immunokits of Biomerieux France, based on the principle of single radial immunodiffusion (28,29). After placing 5 ul of serum samples on each well on plates (IgG IgA IgM) as well as C 3 and C 4 the plates were incubated for 72 hours for IgG, IgA, C3 and C4 and 96 hours for IgM at room temperature. At the end of this period, the diameter of precipitation was measured and converted mg/dl units using a table supplied by the manufacturer.

Isolation of P. mirabilis
Out of 283 clinical specimens of UTI sources, 51 isolates (18.02%) were identified as P. mirabilis. The percentages of P. mirabilis isolated from various clinical specimens are presented in Figure 2.

Identification of P. mirabilis isolates
The isolates were first identified as related to the genus P. mirabilis by swarming phenomenon on blood agar, the characteristic smell of cultures. Microscopic examination of the bacteria appeared as straight rods and gram-negative when it was stained with Gram stain. Several conventional biochemical tests were done to characterize the suspected P. mirabilis isolates. All the 51 isolates of P. mirabilis were confirmed with VITEK II compact system.

Molecular characterization of P. mirabilis isolates
All isolates of P. mirabilis underwent a PCR assay to confirm these isolates' identity, using the ureR gene to detect P. mirabilis. Results of molecular identification shown in Figure 3 indicated that all P. mirabilis isolates were positive for the presence of ureR gene at 225 bp amplicon size. Generally, all 51 isolates of P. mirabilis (100%) exhibited positive PCR products on gel electrophoresis for the ureR gene at 225 bp.

Antibiotic susceptibility patterns of P. mirabilis isolates
The susceptibility patterns of the P. mirabilis isolates   are presented in Table 2. It was found that the more effective antibiotic against isolates was the Meropenem, where all isolates of P. mirabilis were sensitive to it. Adversely, the less effective antibiotic was tetracycline when all isolates resisted them. However, the effect of other antibiotics was variable among the P. mirabilis isolates.
Antibiotic resistance profile revealed that a vast resistance was generally detected among the P. mirabilis isolates against the antibiotics used. It was found that out of 51 isolates of P. mirabilis isolates, 100% resistance property was found to aztreonam and tetracycline, more than 90% resistance to cefepime, ciprofloxacin, imipenem, and piperacillin; and about 70% or less resistance pattern was identified to amikacin, gentamicin, levofloxacin, netilmicin, and tobramycin. Variable degrees of resistance by (6)

Reducing the power of AgNPs and extracts
Reducing power assay is a new approach used to analyze various medicinal plants to detect anti-oxidant activities and reduce Fe 3+ to Fe 2+ . This is because anti-oxidants are potent agents of reduction. Many researchers have also documented that the decrease of bioactive compounds is linked to antioxidant activity (30). The anti-oxidant activity of the clove, black cumin seeds, and ascorbic acid was measured by FRAP assay. Extracts and AgNPs of both clove and black cumin seeds indicate that they are the most effective electron donor and can reduce the oxidized intermediates highly reactive molecules like free radicals and reactive oxygen species of peroxidation processes and the FRAP value was 155.7142 µM, 150.6731 µM for clove and black cumin seeds extract when compared with ascorbic acid. Ascorbic acid is usually used as a standard for preparing the standard curve and has high antioxidant activity (FRAP value = 122.891 µM). Hence, it is essential to determine the reducing power of phenolic constituents to explain the relationship between their anti-oxidant effects (Table 3).

UV-Visible spectra of AgNPs
The UV-Vis spectra were captured of AgNPs with extracts, where distilled water was used as a blank. As illustrated in Figure 4, the absorbance peak was observed at 450 nm. Increased intensity is due to the excitation of surface plasmon resonance (SPR) in the AgNPs, implying the formation of silver nanoparticles. Because the synthesis environment, as well as the shape and size of the nanoparticles, affect the rate of SPR adsorption, as the concentration of nanoparticles increases, the plasmon bands expand and show an absorption tail. This phenomenon indicates an increase in the size distribution of nanoparticles. Previous studies have reported the effect of silver nanoparticles on the formation of  adsorption bands at 400 nm. The SPR band for silver nanoparticles has also been reported at around 435 nm (31) (Figure 4). (32) reported that the color change of the synthesis mixture (herbal extract and silver nitrate as substrate) represents the formation of silver nanoparticles. This agrees with the results of (33) who remarked the color change as an indicator of nanoparticle formation. Surface-active particles have been proposed by (34) as crucial agents in the formation of silver nanoparticles. As a result of electrostatic interactions, functional agents contained in herbal extract interact with silver ions and transform them into nanoparticles. Some of the functional agents in herbal extract involved in the formation of nanoparticles include Flavones, amides, carboxylic acid, aldehydes, ketones, terpenoids, quinines, and anthraquinones (35).

Fourier transform infrared spectroscopy analysis
FTIR analysis was carried out for the identification of possible biomolecules present in the plant extract responsible for the reduction of Ag+ into Ag o . FTIR spectra of AgNPs and plant extracts are present in Figure  5. The spectrum of AgNP-Black seed shows different main peak positioned at 1406 cm -1 , 1467 cm -1 , 1517 cm -1 , 1658 cm -1 , 2850 cm -1 , 2922 cm -1 and 3414 cm -1 . The similarities between (a) AgNP-Black seed and (b) Extract spectrums, few peaks indicate the presence of plant extract in the sample. The intense and wide peak that appeared at 3414 cm -1 denotes the presence of hydrogen-bonded N−H stretching vibrations of amide groups respectively (36). The peak at 2922 cm-1 may belong to the C−H stretching of the alkane group. The bands appearing in the range of 1700-1600 cm -1 and 1100-1000 cm -1 denote the C=O and C−O stretching vibrations, respectively. According to the previous study, the band appeared in the ranges of 1700-1600 cm -1 in the spectrum (a) indicates the formation of AgNP-Black seed capped with different bio moieties (37). The peak located at 1095 cm -1 could be assigned to the C−O stretching of vibration of carboxylic groups. The peak at 717 cm -1 is attributed to aromatic groups. These observations confirmed the presence of different biomolecules which might act as reducing and stabilizing agents during the synthesis of AgNP-Black seed (38).
FTIR results of AgNP-Clove indicate that absorption bands at 3415 (O-H stretching, H-bonded of alcohols, phenols, and N-H stretching of primary, secondary amines, amides of protein), 1600 (C=C stretching and N-H bend of alkenes and primary amines), 1384 (C-C stretching of aromatics) and 1016 (C-O stretching of alcohols, carboxylic acids, esters and ethers and C-N stretching of aliphatic amines). So, it is assumed that these biomolecules and some proteins are responsible for capping, stabilization, and reduction of Ag + to AgNPs. The FTIR analysis indicated the involvement of amides, alkanes, carboxyl, alcohols, and phenols group presented in the synthesized AgNP-Clove (20).

Antimicrobial activities of extracts and AgNPs
The antibacterial activity of extracts and AgNPs produced from this plant was evaluated against P. mirabilis using agar well diffusion and MICs. As shown in Tables (4), synthesized AgNPs are more effective against the negatively charged bacterial cell than the positively charged bacterial cell. Some works showed   that the formed AgNPs using plant extract were more toxic against positive pathogens than negative ones. By increasing AgNPs and plant extract concentration, the inhibition zone diameter also increased. In the case of plant extract, by decreasing in concentrations, no inhibitory was observed. It might be that the AgNPs had better penetration through the agar and consequently into cell bacteria than the plant extract due to the small size of nanoparticles. The highest inhibition zone diameter was achieved for P. mirabilis (35 mm and 20 mm) for AgNP-Clove and clove extract at 512 μg. mL -1 . While the minimum inhibition zone diameter was recorded at 22 mm at 128 μg. mL -1 and 5 mm at 256 μg. mL -1 for AgNP-Clove and clove extract, respectively. However, the highest inhibition zone of both black seed and AgNP-Black cumin seed was recorded at 512 and 256 μg. mL -1 was 23 mm and 7 mm, respectively. And, the lowest inhibition zone was found at 64 μg. mL -1 . The MIC value was performed for P. mirabilis, demonstrating that the AgNPs have the maximum antibacterial efficacy to this microorganism ( Table 5). The MIC of clove extract and C-AgNPs was 512 and 128 μg. mL -1 respectively. However, the value of MIC of both black cumin seed extract and B-AgNPs were 512 and 256 μg. mL -1 respectively.

Extracts and AgNPs
Samuggam et al (39) performed the antimicrobial activity of silver nanoparticles (AgNPs) synthesis from S. mombin leaf extract with disc diffusion method. For the positive and negative control, ciprofloxacin commercial antibiotic disc and 10% DMSO were used, respectively. They found that the antimicrobial activity of ethanolic extract of S. mombin and AgNPs against P. mirabilis. The diameter of the inhibition zone of AgNPs is higher (21 mm) when compared with plant extract (9.33±0.33). Also, (40) were conducted the antibacterial activities of bio(AgNPs) toward P. mirabilis, this value reached 50 μg. mL -1 . For P. mirabilis, the AgNPs exhibited the lowest MBC at 100 μg. mL -1 . On the other hand, the well-diffusion method was assessed and was shown the highest antimicrobial activity against P. mirabilis (10 mm), when they were compared with both positive and negative control (25 and 2 mm, respectively).
The antimicrobial properties of silver nanoparticles are due to their penetration into the cell, binding to cell membrane receptors and membrane destruction, disruption of the process of intracellular energy production and release of cell sap (41). In fact, adhesion and destruction of the cell wall are one of the mechanisms of inhibition of gram-negative bacteria by silver nanoparticles (42). The production of free radicals by silver nanoparticles, which destroy cell membranes, is another antibacterial mechanism of these nanoparticles. Due to the interaction of silver ions with thiol groups, the interaction of silver nanoparticles and DNA prevents cell division and DNA replication, which ultimately leads to cell degradation (43).
Inhibition of protein and nucleic acid synthesis, as well as cell membrane degradation, is the common mechanism of nanoparticles and antibiotics to inhibit bacterial growth (44). In addition, there are several specific inhibitory mechanisms of silver nanoparticles: [1]: the attachment of nanoparticles to cell membranes and the creation of pores in them that disrupt membrane permeability and the release of intracellular contents (45); [2] the production of free radicals by silver nanoparticles that inactivate and destroy DNA molecules; [3]: Release of silver ions due to interaction between silver nanoparticles and thiol groups of bacterial proteins that inactivate many key bacterial proteins (46); [4]: Disruption of the ATP production process (47). Bacterial cells must undergo several gene mutations to counteract these effects, which is unlikely. On the other hand, P. mirabilis. genome carries antibiotic resistance loci towards different mechanisms of antimicrobial agents and metals, including swarming mobility, biofilm formation, enzymatic detoxification, and efflux systems by employing the PATRIC and PGAAP gene explanation services (48).

Inhibition of swarming activity of P. mirabilis
The swarming parameters expressed in terms of the number of concentric rings, diameters of swarm rings were determined to be reduced by different extracts and AgNPs concentrations. The pronounced reduction was observed in the colony diameter by extracts and AgNPs, in concentrations ranging from 32-512 mg. mL -1 . The negative control plate showed seven concentric rings. The number of rings was reduced to 1 in the medium, having 64 mg. mL -1 and 512 mg. mL -1 of clove extract and AgNPs-Clove, but in the black seed extract and AgNP-Black cumin seed, the ring numbers were reduced to 0 at 256 mg. mL -1 and 1 at 512 mg. mL -1 , respectively (Table 6).
Saleh et al. (49) reported different swarming behavior for isolates. The range of P. mirabilis isolates is between 21 and 42 mm. It has been observed that silver nanoparticles in sub-MIC concentrations prevent P. mirabilis from increasing in diameter. Our results are consistent with those reported by (50). The author showed that the production of a large number of virulence factors by P. mirabilis, such as hemolysin and protease, exacerbates infectious diseases. These virulence factors play an important role in increasing the motility of swarming cells and their movement from the urinary tract to other parts of the host tissue. Although not many studies have been done on the effect of silver nanoparticles on swarming cell formation; in some previous studies, the effect of nanoparticles on the rhl regulatory system that controls swarming motion has been mentioned (51). The results indicate the negative effect of silver nanoparticles on swarming behavior. It has also been shown that silver nanoparticles reduce swarming mobility by suppressing The swarming movement was the reason of combination sensory transduction and universal control operations. The swarming cells called for sensing and coupling of an assortment of cell-to-cell, environmental and intracellular signals, and involved adjusting the expression of gene networks key to physiological and morphological alterations. Alternations in gene flaA encoding flagellin protein and flhDC gene contributed to the upregulation of flagellin protein production, C and wad genes needed for the core region of LPS (53). Other genes implicated in flagellum construction drove the repression of Proteus rods' segregation and inhibition of swarming (49).

Biofilm formation assay
The biofilm mode of growth confers on the associated organisms a measurable decrease in antimicrobial susceptibility; for this reason, it is essential to know the times of growth and biofilm development. The ability of the strains to form biofilms on abiotic surfaces was evaluated by the crystal violet test. In this assay, all P. mirabilis isolates showed the ability to produce biofilms. Biofilm formation capability in these isolates was classified into three groups, strong biofilm producers (17.65%), moderate biofilm producers (39.22%), weak biofilm producers (27.45%), and non-biofilm former (15.69%) ( Table 7). (6) were conducted an MTP assay of 176 isolates of P. mirabilis isolates, 162 (92.05%) were biofilm producers and 14 (7.95%) were non-producers. Of the biofilm producers (n = 162), 78 (48.15%) were moderate biofilm producers, whereas 62 (38.27%) and 22 (13.58%) were strong and weak biofilm producers, respectively. Similarly, (54) tested strains of P. mirabilis showed variable results in the aspect of biofilm formation. Most clinical strains were put into the strong biofilm-former category (75, 20 and 5% were strong, moderate, and weak biofilm-formers, respectively). Two isolates from the strongest biofilm-forming P. mirabilis strains were selected for biofilm inhibition studies.

Immunological assessments of UTI patients
The serum from patients with UTI was tested for the presence of immunoglobulins (IgA, IgG, and IgM) and complement components (C3 and C4) by immunodiffusion plates.    Figure 7 A, B).

Discussion
Bacteria, commensals as well as pathogens, are constantly surrounding the human body, frequently as members of the microbiota. The early defense against bacterial infections depending on the various mechanisms of the innate and adaptive immune response, in which cytokines play a crucial role to initiate the inflammatory response (55,56). The innate and adaptive immune systems are responsible for preventing pathogenic bacteria from penetrating either by the protective barrier of the skin and mucous membranes or through the humoral and cellular immune response (57).
Immunoglobulins are a group of serum proteins with a crucial antimicrobial activity, IgM represents an indicator of recent infection, IgG levels increased in chronic infections, while IgA contains a secretory part that helps in reducing the mucosal and secretory based infections (58). The humoral immune response to antigenic or inflammatory challenges, such as infection either bacterial or other microorganisms, vaccination or trauma, has been proposed to comprise the polyclonal activation of memory B cells of unrelated specificity (59).
The results of this study showed a significant increase in the mean level of serum IgG and IgA (P ≤ 0.001) in UTI patients compared to the control group, while the level of IgM was elevated in UTI patients but showed no significant differences (p-value ˃ 0.001). Serum antibodies found in UTI-positive patients have bactericidal activity (60). Antibodies in urine may resist UTI by preventing the adherence of bacteria to uroepithelial cells, the role of humoral immune response which increased IgG and IgA, IgM production were associated with UTI (61). Also, bacteria are killed by infected human serum through the lytic activity of the complement system. The results of the current study were in agreement with (61) and (62) who found that there were increases in the levels of immunoglobulins in UTI patients compared with the control group, also with the study of (63) and (64) who showed that E. coli was the major pathogen of UTI followed by Proteus spp, and they found that IgG and IgA concentrations significantly increased in UTI patients in comparison to control group while IgM concentration in the study of (63) was decreased in UTI patients. While it disagreed with the results of (65). This variation in results was might be due to the difference in study conditions and social status of study patients.
Elevation in serum concentration of IgG in patients suffering from UTI may be the reason for the significant increases in the concentration of complement components in patients suffering from UTI (64).
Increasing in serum mean levels of antibodies may be due to activation of B cell by antigenic stimulation that causes B cell to divide and differentiate into an antibody-producing cell called a plasma cell. Induction of production-specific antibody without the assist of T lymphocytes for example large molecular weight antigen with regular repeating epitopes involve Pneumococcal polysaccharide, flagellar and fimbrial antigen is IgM as a major antibody, this type of antigen called T cell-independent antigen (66). Some bacterial antigens can't induce B cells directly for producing immunoglobulins but by helping T lymphocytes called T cell-dependent antigen, the switch to other isotypes for example IgA and IgG, production needs the presence of cytokines and other signals secreted by locally responding T cells (58,66). Increased values of IgA in sera of UTI patients was due to immune reaction against causative agents of    infection, this result agreed with the findings of (61). Urinary tract infection patients were showing more IgM values in sera than control individuals. Increased IgM values in patients groups were attributed to the surveillance of the immune system in the body and reaction against infection. The IgM undergoes first release within the primary immune reaction in acute inflammation to combat infection (66,67). This result agreed with the findings of (64,68). While it disagreed with the results of Azat, 2012 (65). This variation in results was might be due to the difference in study conditions and social status of study patients.
The hypergammaglobulinemia found in patients with UTI may reflect specific as well as non-specific polyclonal activation of B-lymphocytes. Many bacterial species are known to be capable of non-specific stimulation of B-lymphocytes and cause polyclonal B-cell activation, DNA-synthesis and proliferation and immunoglobulin synthesis (69). The quantitative estimation of total serum immunoglobulin in all classes may therefore help to make the differential diagnosis of whether a bacterial infection is chronic or acute.
Complement components C3 and C4 were increased in UTI patients in comparison to control groups. These results agreed with that reported by others (68) and disagree with the results of (70). Several studies have been conducted to assess the role of C3 in infectious diseases (70)(71)(72)(73). The increasing of complement component C3 in the blood circulation of patients with UTI may return to the production of complement proteins by hepatocytes and several extrahepatic tissues that involved glomerular epithelial cells, endothelial cells, mesangial cells and human proximal tubular epithelial cells (74,75). The C3b fragment which produced by C3 split binds to fimbriae expressing on uropathogenic then C3b binds with Decay accelerating factor (CD55) has been located on the surface of human renal epithelial cells (74), CD46 can act as a human epithelial cell receptor for entering of opsonized uropathogenic E coli (76).
Infection, in general, caused an increase in the C3 component of the complement because this component is activated in classical and alternative pathways, while the elevation of the C4 component of the complement has occurred in the classical pathway more than an alternative pathway. UTI infection is a mixed infection, it causes by different types of bacteria some of them producing Exo-Toxins while others producing Endo-Toxins, so alternative pathways and classical pathways may be activated when the bacterial toxin is produced by bacteria while the classical pathway is activated by bacteria itself (68). Complement components have an important role in Inflammatory processes UTI infection attributed the increase of complement component production to the stimulation of the inflammatory mediators (77).
In addition, the C3 complement represents a critical component of the complement cascade, subserves several critical functions in human immune response and enhances bacterial killing and its levels correlate with infectious diseases (70). In addition, recurrent UTI is believed to be associated with the host immune system; Complement cascade activation is an effective immune mechanism that occurs in three different forms (the classic, alternative, and mannan-binding lectin) and is activated by pathogens. Regardless of the pathway, this activation causes the formation of C3 and C5 convertases, which in turn increases the production of bioactive compounds such as anaphylatoxins, opsonins, and membrane attack complex (70). Surrounding cells and recruited inflammatory cells then take the decisions about the quality and quantity of the mucosal response, leading to disease or protection. The epithelial cells are thus important players in the mucosal immune system; acting both as docking sites for the bacteria and as sensors of microbial attack. So far, several studies have been conducted on the effect of nanoparticles in biological fields (78)(79)(80)(81)(82)(83)(84)(85). In this regard, it is better to investigate the relationship between the use of nanoparticles with some abnormalities and complications such as the effect on gene polymorphism (86), mitochondrial disorders (87), sexual disorders (88) and overall human health (89).